Time-resolved ligand dynamics revealed in a β-lactamase using room-temperature serial crystallography

Abstract β-Lactamases hydrolyse β-lactam antibiotics and are key antimicrobial resistance determinants in globally important bacterial pathogens. Here, we report on the use of time-resolved room-temperature drop-on-fixed-target serial crystallography, complemented by classical single-crystal crystallography and molecular simulations, to investigate reversible covalent inhibition of the serine β-lactamase CTX-M-15 by the diazabicyclooctane inhibitor avibactam, used to treat infections by β-lactamase producing organisms. A covalent ring-opened avibactam carbamoyl complex is observed in crystal structures 80 ms after mixing, which rearranges on a 1.3 s to 600 s time scale. Together with molecular dynamics simulations and density functional theory calculations based on our experimental structures, our data suggest that in the initially observed species the avibactam N-sulfate nitrogen is poorly positioned to attack the carbamoyl carbon to initiate the 5-exo-trig recyclisation required for inhibitor reformation. This complex then undergoes conformational changes that potentially prime it for this intramolecular reaction. These data reveal the conformational dynamics and stereoelectronic basis of diazobicyclooctane inhibition and demonstrate how the combination of room-temperature serial crystallography and molecular simulations can capture ligand motions in enzyme active sites on millisecond-to-second timescales, delivering mechanistically relevant understanding of the dynamic behaviour of enzyme-bound species.